Cell type-specificity elements of the immunoglobulin heavy chain gene enhancer.

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RESUMO

A strong transcriptional enhancer was created by oligomerization of a short segment from the immunoglobulin heavy chain (IgH) enhancer. This segment was analyzed in parallel for biological activity in vivo and factor binding in vitro. In transfection experiments the oligomerized segment stimulates transcription in a cell type-specific manner similar to the entire IgH enhancer. Transfections of mutants identified two sequence motifs whose integrity is required for efficient and cell type-specific activity of this enhancer. The first is a sequence suggested previously to be bound by a factor in vivo, and the second is a highly conserved decanucleotide which also occurs in Ig variable gene promoters. The ability of these two sequence motifs to bind proteins in vitro was tested by band shift assays. Under our in vitro conditions we could not detect proteins binding to the in vivo footprint region. However, we found protein factors binding to the decanucleotide. A ubiquitous form of this factor is present in every cell line analyzed. Additional variants are detected exclusively in cells where the IgH enhancer and the segment thereof are active. Elimination of the decanucleotide motif is not only a strong down mutation in vivo but also abolishes binding of all factor variants in vitro. Thus our data suggest that the two enhancer motifs analyzed are involved in positive rather than negative control of transcription.

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